Viruses are obligate parasites that use the host's mechanisms for expressing their genes and propagating themselves. Bacteriophage, or “phage” for short, are viruses that infect bacteria. Phage have a host range that is often fairly narrow, infecting a single species or a group of related species.
Some bacteriophage, such as T4, always kill their hosts within a short time after infection—they are called LYTIC phages. Others, such as Lambda, can also establish themselves in a dormant form within the cell and be maintained stably over many generations—they are called LYSOGENIC phages. On infection lysogenic phages have a choice between a lytic cycle or to establish lysogeny.
In a lysogenic cycle, the phage integrates into the host chromosome at a specific point using a site-specific recombination process. The expression of most phage genes is shut down by a repressor and the phage genome can be replicated as part of the host chromosome. Under certain conditions, usually involving host stress, the genes get switched on again and the phage genome is excised from the chromosome, replicated and new phage particles are released by lysing the cell.
After their discovery early in the 20th century, phage were widely used to treat various bacterial diseases in people and animals. After an enthusiastic beginning, poorly designed research protocols and the development of antibiotics ended most phage therapy research in the West. However, phage therapy continued in Poland and the former Soviet Union for decades and the Eastern European experience should serve to bootstrap the technology into the U.S. Thus, although there are few, if any, clinical trials underway in the U.S., the technology is reasonably well developed and its advantages and limitations are well understood.
The alarming rise in antibiotic resistance in bacteria is leading to a resurgence of interest in bacteriophage research. In fact several companies exist that are dedicated to the realization of phage therapy. The Eliava Institute of Bacteriophage, Microbiology & Virology in the Republic of Georgia has been an international leader in phage therapy for over 70 years and has the world's largest collection of therapeutic phages. Other companies including Exponential Biotherapies, Inc. of New York; Intralytix, Inc. in Maryland; Phage Therapeutics International, Inc. (PhageTx) of British Columbia; Biophage, Inc. of Quebec; and PhageTech, Inc. of Quebec are developing commercial phage therapies.
The limitations and advantages of phage therapy are listed by Elizabeth Kutter of Evergreen State College at www.evergreen.edu/phage/phagetherapy.html. Her lists are worth iterating here in order to understand the potential and limitations of phage therapy. The limitations include: 1) Paucity of understanding of the heterogeneity and ecology of both the phages and the bacteria involved; 2) Failure to select phages of high virulence against the target bacteria before using them in patients; 3) Use of single phages in infections that involve mixtures of different bacteria; 4) Emergence of resistant bacterial strains, which can occur by selection of resistant mutants (a frequent occurrence if only one phage strain is used against a particular bacterium) or by lysogenization (if temperate phages are used); 5) Failure to appropriately characterize or titer phage preparations, some of which were totally inactive; 6) Failure to neutralize gastric pH prior to oral phage administration; 7) Inactivation of phages by both specific and nonspecific factors in body fluids; 8) Liberation of endotoxins as a consequence of widespread lysis of bacteria in the body (this is called the Herxheimer reaction); and 9) Failure to identify the bacterial pathogens involved necessitated by the relative specificity of phage therapy.
The advantages of phage therapy include: 1) They are self-replicating and self-limiting, because they multiply only as long as sensitive bacteria are present and then are gradually eliminated; 2) They target specific bacteria, causing less damage to the normal microbial balance in the body; 3) Phage can often be targeted to receptors on the bacterial surface that are involved in pathogenesis, so that any resistant mutants are attenuated in virulence; 4) Few side effects have been reported for phage therapy; 5) Phage therapy would be particularly useful for people with allergies to antibiotics; 6) Appropriately selected phages can easily be used to prevent bacterial disease in people or animals at times of exposure, or to sanitize hospitals and help protect against hospital-acquired infections; 7) Phage can be prepared fairly inexpensively and locally, facilitating their potential applications to underserved populations; 8) Phage can be used independently or in conjunction with other antibiotics to help reduce the development of bacterial resistance; and 9) Multiple delivery means are available, including noninvasive means like topical application, oral administration, and inhalation.
One lytic phage of long time interest to our laboratory is called “SPO1.” SPO1 has a linear dsDNA genome of 140 kb, and its host is the bacterium Bacillus subtilis. Early in SPO1 infection of B. subtilis, the synthesis of most host-specific molecules is replaced by the corresponding phage-specific biosynthesis. Subversion of the host machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal repeat in an 11.5-kb “host-takeover module.” The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12.
The 24 genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified therein. Transcription-termination and RNase III cleavage sites were identified in the nucleotide sequence as well. We have placed most of these 24 genes into an inducible B. subtilis/E. coli shuttle vector and introduce them here for use in phage therapy and as antimicrobial agents.